Phenylketonuria (PKU) is a metabolic disorder that results from impaired activity of hepatic phenylalanine hydroxylase (PAH), the enzyme responsible for disposal of the majority of phenylalanine intake. Currently, the only known treatment for classical PKU is a very difficult to maintain diet for life. Development of a therapeutic treatment would assist the current dietary treatment and prevent the neurological damages inflicted on those individuals with PKU, particularly for those patients with the most severe forms of the disease. This proposal combines the basic PKU research efforts of the Stevens laboratory at The Scripps Research Institute with the clinical PKU research efforts of the Scriver laboratory at the McGill University and Montreal Children's Hospital. Based on our recent success in developing a less immunogenic injectable PKU enzyme substitute using pegylated phenylalanine ammonia-lyase (PEG-PAL; reported by Gamez, et al. (2005) Mo/. 77?er.,11;986-989 and Wang, et al. (2005) Mol. Genet. Metab. 86:134-140), this proposal details the development of a structure-based PKU enzyme substitution therapy using modified PAL or pegylated variations of modified PAL (for stability, protease resistance, and reduced immunogenicity) to create an oral therapeutic. The specific objectives of this study are to use structure- based molecular engineering to optimize a form of PAL with improved characteristics for potential use in PKU oral therapy (Aim 1). Further optimization of PAL will incorporate pegylation procedures to develop a form of the enzyme for PKU oral therapy use (Aim 2). Animal studies will be conducted starting in the first year on the most promising modified PAL candidates, and PK/PD/ADME/toxicity studies will be completed by the end of the funding period, making a favorable orally available therapeutic PAL molecule available for human trials (Aim 3). [unreadable] [unreadable] [unreadable]